Figure 5. ATP-depletion traps ESCRT-dependent Ypq1 sorting intermediates.
(A) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Vph1-mCherry. Images show untreated cells (top), Rapamycin (1 μg/ml, pretreated for 1 min) and NaN3/NaF (10 mM each, final concentration) treated cells (middle), and cells treated with only NaN3/NaF. All cells were grown at 30°C (bottom). (B) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Vph1-mCherry. Images shows cells treated with Rapamycin and NaN3/NaF for 30 min (top), and cells from the same culture that have been washed with water and re-suspended in fresh YPD medium (30 min after wash) (bottom). (C) Western blot analysis of Ypq1-FKBP degradation. Wild-type cells were treated with rapamycin, with or without NaN3/NaF. After washing with deionized water and resuspended in fresh YPD medium, the NaN3/NaF-treated cells were collected at 15 min and 30 min. Cells were grown at 30°C. The blot was probed with GFP and G6PDH antibodies. (D) Fluorescent microscopy analysis of Ypq1-FKBP in wild-type and vps27Δ cells. Images show cells before and 15 min after rapamycin and NaN3/NaF treatment. Scale bars represent 2 μm.